Despite the identification of tumor-associated antigens in various malignancies, the vast majority of the antigens are at best weakly immunogenic. Explanations for this include preexisting tolerance to self-antigens, poor antigen presentation by tumor cells, and immune inhibition by mediators secreted by tumors (e.g. vascular endothelial growth factor and IL-10). Dendritic cells (DC), which are extremely efficient antigen presenting cells (APC) uniquely capable of sensitizing nave T cells to antigen, have been used to reverse this immunologic unresponsiveness. Armed with tumor associated antigens, DC are capable of priming tumor specific immune responses in vitro and in vivo, often leading to tumor protection in various animal model systems. Recent clinical trials using DC have demonstrated that they can induce T cell and B cell immune responses against tumor associated antigens. Moreover, clinical responses have been observed in some patients. Representing less than 1 percent of circulating white blood cells, modest numbers of DC can be obtained from human peripheral blood by several techniques including density centrifugation and in vitro culture with exogenous cytokines. Advances making DC-based vaccination more potent and less complex will be required if this immunotherapeutic approach is to succeed clinically. The current proposal seeks to address several fundamental issues in DC immunotherapy in patients with lung and colorectal tumors with a peptide derived from carcinoembryonic antigen (CEA) as the immunologic target. We will explore the ability of a novel modified CEA peptide, with an amino acid substitution making it more immunogenic, to help break tolerance to this self-antigen. We will also examine approaches to increase tumor antigen delivery into DC via a targeting peptide. We will develop in vitro assays to evaluate the immunologic efficacy of these vaccine strategies. Finally, we will investigate the ability of a novel cytokine Flt3L to expand DC in vivo for DC harvesting in a clinical trial. The proposed studies will provide the foundation for simplified yet more potent DC targeted cancer immunotherapy for the future.